Deterred but not preferredPredation by native whelk Reishia clavigera on invasive bivalvesAstudillo Juan C Bonebrake Timothy C Leung Kenneth M Y

Published inPLoS ONE

Online published 16052018

Document VersionFinal Published version also known as Publisherrsquos PDF Publisherrsquos Final version or Version of Record

LicenseCC BY

Publication record in CityU ScholarsGo to record

Published version (DOI)101371journalpone0196578

Publication detailsAstudillo J C Bonebrake T C amp Leung K M Y (2018) Deterred but not preferred Predation by nativewhelk Reishia clavigera on invasive bivalves PLoS ONE 13(5) httpsdoiorg101371journalpone0196578

Citing this paperPlease note that where the full-text provided on CityU Scholars is the Post-print version (also known as Accepted AuthorManuscript Peer-reviewed or Author Final version) it may differ from the Final Published version When citing ensure thatyou check and use the publishers definitive version for pagination and other details

General rightsCopyright for the publications made accessible via the CityU Scholars portal is retained by the author(s) andor othercopyright owners and it is a condition of accessing these publications that users recognise and abide by the legalrequirements associated with these rights Users may not further distribute the material or use it for any profit-making activityor commercial gainPublisher permissionPermission for previously published items are in accordance with publishers copyright policies sourced from the SHERPARoMEO database Links to full text versions (either Published or Post-print) are only available if corresponding publishersallow open access

Take down policyContact lbscholarscityueduhk if you believe that this document breaches copyright and provide us with details We willremove access to the work immediately and investigate your claim

Download date 10012021

RESEARCH ARTICLE

Deterred but not preferred Predation by

native whelk Reishia clavigera on invasive

bivalves

Juan C Astudillo12 Timothy C Bonebrake2 Kenneth M Y Leung123

1 The Swire Institute of Marine Science Faculty of Science The University of Hong Kong Shek O Hong

Kong China 2 School of Biological Sciences The University of Hong Kong Hong Kong China 3 State Key

Laboratory in Marine Pollution City University of Hong Kong Kowloon Hong Kong China

jcarlosconnecthkuhk (JCA) kmyleunghkuhk (KMYL)

Abstract

This study tested the potential bio-control role of the common native predatory whelk Reishia

clavigera on the invasive bivalves Xenostrobus securis and Mytilopsis sallei and the native

Brachidontes variabilis in Hong Kong Predation experiments were conducted in the labora-

tory under salinity levels of 22permil and 32permil as well as under field conditions The results indi-

cate that the invasive bivalves are more vulnerable to predation than the native bivalve in

environments with high salinity whereas environments with moderately low salinity (22permil)

may reduce predation Because R clavigera did not show clear prey preference the low sur-

vival of the invasive species might be due to a lack of effective anti-predatory defenses under

experimental conditions These findings could explain the high abundance of the invasive

bivalves in disturbed environments in Hong Kong where predation appears to be lower

Introduction

Introduced species can establish and become invasive in novel environments if they are able to

escape from natural enemies (predators competitors and parasites) that would otherwise

limit their distribution and abundance [1ndash3] Invasive species may find a spatial niche within

native communities under unfavorable environmental conditions [4ndash6] Similarly estuaries

and disturbed bays are known to be more susceptible to invasions than open and less disturbed

coasts [7 8] Therefore understanding the interactions between native predators and the envi-

ronmental conditions on the survival of invasive species could provide insights into the control

of their abundance and spread [9 10]

Gastropods (grazer and predatory) are recognized for their strong impacts structuring ben-

thic communities [11] Few species have been tested as potential biological control agents on

fouling communities that could eventually reduce the abundance and spread of invasive spe-

cies [12] Predatory gastropods from the subfamily Rapaninae present high diversity in the

Indo-West Pacific [13 14] In the Northwestern Pacific Reishia clavigera (Kuster 1860 for-

merly named as Thais clavigera) is one of the most common and abundant species occurring

in rocky shores and fouling communities from habitats with moderately low salinity to oceanic

PLOS ONE | httpsdoiorg101371journalpone0196578 May 16 2018 1 16

a1111111111

a1111111111

a1111111111

a1111111111

a1111111111

OPENACCESS

Citation Astudillo JC Bonebrake TC Leung KMY

(2018) Deterred but not preferred Predation by

native whelk Reishia clavigera on invasive bivalves

PLoS ONE 13(5) e0196578 httpsdoiorg

101371journalpone0196578

Editor Geerat J Vermeij University of California

UNITED STATES

Received November 6 2017

Accepted April 16 2018

Published May 16 2018

Copyright copy 2018 Astudillo et al This is an open

access article distributed under the terms of the

Creative Commons Attribution License which

permits unrestricted use distribution and

reproduction in any medium provided the original

author and source are credited

Data Availability Statement All the individual data

points behind the results tables and figures are

publicly available at Open Sciences Framework

httpsosfiofazk9 DOI 1017605OSFIOCTGK2

Funding This study was partially supported by the

Environment and Conservation Fund (ECF Project

2010-28) of the Hong Kong Special Administration

Region Government There was no additional

external funding received for this study

Competing interests The authors have declared

that no competing interests exist

conditions [15ndash17] In natural habitats R clavigera is an important predatory species structur-

ing intertidal communities where it preys on a wide number of invertebrates such as bivalves

limpets chitons barnacles and serpulid polychaetes [18ndash20] Reishia clavigera preys upon

bivalves by boring through the bivalve shell (chemo-mechanical process) [16 17] The high

dietary plasticity of R clavigera which can change according to its ontogenetic stage andor to

spatial and temporal prey availability [17] suggests that it could have an important role in con-

trolling the spread of invasive invertebrates

In Hong Kong the introduced bivalves Xenostrobus securis (Lamarck 1819) and Mytilopsissallei (Recluz 1849) have become invasive only in some estuarine fouling communities [21

22] A previous study indicated that these invasive bivalves tolerate a greater range of salinities

than the native counterpart Brachidontes variabilis (Krauss 1848) which could explain their

dominance under estuarine conditions [23] The wide environmental tolerance of these inva-

sive species suggests that they have the ability to spread in habitats with moderately low salinity

and oceanic conditions In the 1980rsquos M sallei dominated fouling communities under oceanic

conditions [24] but it is currently restricted to estuarine and disturbed environments [25]

The invasion of X securis in estuaries in Europe could be favored by low predation compared

to a native mussel [26] In Hong Kong several studies have demonstrated that predatory gas-

tropods play an important role on bivalve communities [17 19 27] however their potential

role in predation on invasive bivalves remains undetermined

The aim of this study was to compare the survival of the invasive bivalves Xenostrobussecuris and Mytilopsis sallei and the native Brachidontes variabilis exposed to the native whelk

Reishia clavigera under laboratory conditions with either normal or moderately low salinity as

well as field conditions Field experiments also tested the survival of the bivalves to potential

predators located in two fouling communities one pier located in a disturbed site (ie low

salinity and poor water quality) with the absence of R clavigera and another pier under oceanic

conditions with R clavigera This study provides information about the potential role of the

predatory whelk in limiting the spread of the invasive bivalves to other communities and its

use as a biological control agent for the management of invasive bivalves

Materials and methods

Species collection and laboratory conditions

The native whelk Reishia clavigera was collected from the low intertidal and shallow subtidal in

Stanley Pier The invasive bivalves Xenostrobus securis and Mytilopsis sallei were collected from

submerged substrates in the shallow subtidal zone under the pier located in Kwun Tong typhoon

shelter The native bivalve B variabilis was collected from buoys within ~30 cm below the sea

surface in Tai Tam bay (see Fig 1) No specific permissions were required for animal collection

in this study because the species collected for the experiments are not endangered or protected

species and were collected from public piers and buoys that are not protected Xenostrobussecuris and B variabilis both of which belong to the family Mytilidae differ taxonomically at the

subclass level with M sallei which belongs to the family Dreissenidae In the laboratory all spe-

cies were sorted and kept separately in mesh cages in outdoor tanks (under natural photoperiod)

of 100 L with constant seawater supply (sand filtered) and aeration Temperatures and salinities

of the running seawater during the experimental period ranged from 29 to 32˚C and from 31 to

35permil respectively Experimental bivalve individuals ranged from 10 to 15 cm in shell length

whereas the native predatory whelk R clavigera ranged from 25 to 35 cm in shell length This

size range of the bivalves represented the most common size in the field among the three species

The bivalves were fed with the microalgae Chaetoceros gracilis and with commercial coral food

made of phytoplankton (Plancto Aqua Medic Germany) During the acclimation period R

Predation by native whelk Reishia clavigera on invasive bivalves

PLOS ONE | httpsdoiorg101371journalpone0196578 May 16 2018 2 16

clavigera were fed with crushed individuals of the three bivalve species to ensure the predator

recognized all prey species [26] Each individual of the bivalve species and R clavigera were used

only for one experiment of this study to assure independency of the data

Ten individuals of each bivalve species within the range of 10 to 20 cm in shell length

were dried and used to measure the maximum length (L) height (H) and width (W) total

individual biomass (TB) shell biomass (SB) and posterior adductor muscle biomass (AB)

These measurements were used to determine the ratios of H to L W to L AB to TB and shell

thickness index (STI) to describe the morphology of the bivalve species High HL ratios (near

1) indicated round shaped shells high WL ratios indicated cupped shells and high ABTB

ratios indicated that the bivalve had a larger adductor muscle in proportion to the total bio-

mass Higher values for TSI indicated thicker shells TSI was calculated with the equation

described in Fitzer et al (2015) [28]

Laboratory experiments

Based on pilot observations Reishia clavigera preyed on bivalves when there was more than

one whelk per aquarium and the aquarium had developed biofilm on the walls Reishia

Fig 1 Map of the study sites Filled squares indicate where the bivalves were collected and experiments conducted

httpsdoiorg101371journalpone0196578g001

Predation by native whelk Reishia clavigera on invasive bivalves

PLOS ONE | httpsdoiorg101371journalpone0196578 May 16 2018 3 16

clavigera preyed on the bivalves by boring the edge of the shells To ensure that R clavigera fed

on the bivalves the aquaria (35 L) were under experimental conditions with running seawater

for 5 days before starting the experiments for biofilm development Two days before starting

the experiments three individuals of R clavigera were introduced into the predation treatment

aquaria for acclimation without food All the aquaria were covered with plastic mesh (05 x 05

cm) to avoid the escape of the whelks The survival of the invasive and native bivalves exposed

to R clavigera was tested under single choice and multiple choice experiments as recom-

mended by Underwood and Clarke (2005) [29] to determine prey preference by the predator

The single choice experiments provided data for the prey consumption of each species which

was used to estimate the expected proportion of prey species consumed at random in the mul-

tiple choice experiment Prey consumption may vary between species because predators may

catch and handle prey species differently Therefore a preference by the predator would be

demonstrated when the proportion of prey species consumed differed to the expectation

derived from the single choice experiment

Single choice experiment Twelve individuals of a single bivalve species were attached to a

PVC panel (7 x 15 cm) with plastic glue Individuals were carefully glued only by the left valve

to allow the bivalves to open their shells All the panels were kept in tanks with running seawa-

ter for 24-h before the predation experiment to remove any glue residual Each panel with a

single bivalve species was introduced to either the predation treatment (with 3 randomly

selected Reishia clavigera) or the control treatment aquaria (without predator to determine

handling mortality) The experiment was under running seawater and each treatment had 6

replicates (sumn = 2 predation treatments times 3 bivalve species times 6 replicates = 36 experimental

units) After 24-h the number of surviving bivalves in each aquarium was recorded The

bivalves were considered alive when they reacted to the touch on the shell or the valves were

tightly closed Dead bivalves were easily determined because their valves were open with no

flesh whereas few killed bivalves (but not eaten) were open and did not react to touching

Multiple choice experiments Experimental conditions were similar to the single choice

experiment however in this experiment 6 individuals of each bivalve species were randomly

attached to one panel (ie 3 species times 6 individuals = 18 individuals per panel) Panels were

then exposed to the predators (11 replicates each with 3 Reishia clavigera) or to the control

without predators (5 replicates) for 24-h to determine the survival number of the bivalves (ie

16 experimental units) The number of replicates for the control treatment in this experiment

was lower than predation treatment because in the single experiment none of the bivalves in

the control treatment died

Acute temperature and salinity test on Reishia clavigera The survival of R clavigera was

assessed through a 96-h acute temperature and salinity test to examine tolerance before per-

forming the predation experiments under different salinity conditions Reishia clavigera indi-

viduals were acclimated in indoor conditions for two weeks in a 20 L plastic aquarium under

1212 daynight photoperiod filtered seawater (022 μm) with 30 plusmn 2permil and 22 plusmn 1˚C (indoor

room temperature) and constant air supply (via aeration) Every second day whelks were fed

with the bivalve Xenostrobus securis and the seawater was renewed Feeding stopped 24-h

before starting the test Five whelks were placed in a 045 L aquarium Survival was tested

under a combination of three temperatures (14 22 and 30˚C) and three salinities (12 22

32permil) in an orthogonal and balanced design with 3 replicates (sumn = 3 temperatures times 3 salini-

ties times 3 replicates = 27 experimental units) Each aquarium was covered with a transparent

plastic film with small holes and provided with constant air supply using a Pasteur pipette Sea-

water was renewed once at 48-h Survival was monitored every 24-h until the end of the 96-h

experiment Whelks were considered dead when they were not attached and did not react to

the touch of their foot muscle

Predation by native whelk Reishia clavigera on invasive bivalves

PLOS ONE | httpsdoiorg101371journalpone0196578 May 16 2018 4 16

Multiple choice experiments in two salinity conditions A second multiple choice experi-

ment (similar setup to the previous multiple choice experiment) was performed under salini-

ties of 22permil and 32permil Predation of the whelks on the bivalves was tested under these two

salinities because superficial salinity in Kwun Tong typhoon shelter (with high abundance of

invasive species) generally ranges from 195 to 326permil and these two salinities are within the

salinity tolerance range of the bivalves [23] All the aquaria had running seawater for 5 days

before the experiment for biofilm development At 48-h before the experiment the whelks

were introduced to the tank and the salinity was adjusted to the experimental salinity in a step-

wise manner (plusmn2permil per h) Aquaria had constant air supply and seawater was only renewed

every 24-h to keep salinity stable The panels were either exposed to predation by Reishia clavi-gera or to no predation (as control for experimental conditions without predators) under the

two salinity treatments (22 and 32permil) with 6 replicates each (sumn = 2 predation treatments x 2

salinities x 6 replicates = 24 experimental units) The number of bivalve survivors was counted

after 24-h

Field predation experiment

A field predation experiment was conducted in Kwun Tong and Stanley piers to determine

predation under field conditions (Fig 1) These piers were chosen because of the contrasting

conditions fouling communities in Kwun Tong Pier where Reishia clavigera was absent was

dominated by both invasive bivalve species whereas in Stanley Pier where R clavigera was

abundant both invasive bivalve species were absent Preliminarily 20 quadrats (25 times 25 cm)

per pier (low intertidal) were sampled finding that R clavigera was absent in Kwun Tong Pier

whereas in Stanley pier its abundance was on average 504 (SD = 695) individuals per m2 In

the present experiment five individuals of each of the three bivalve species (total 15 individu-

als) were attached to a PVC panel (18 times 18 cm) Panels were randomly used for one of the

three following treatments 1) Predation by R clavigera (bivalves exposed to three individuals

of R clavigera) 2) Open panel (bivalves exposed to any predator occurring in the site) and 3)

control with predators excluded (sumn = 2 sites x 3 treatments x 9 replicates = 45 experimental

units) Predation by R clavigera and the control were achieved by enclosing the panels with

plastic cages (kitchen sieves with mesh of 03 x 22 cm) whereas panels without cages were

exposed to any predator occurring in the study site Panels were randomly attached to the sub-

merged vertical columns (ie pillars) of the piers in the low intertidal zone After 5 days the

panels were collected and the bivalve survival rates were examined and recorded The tempera-

ture and salinity recorded in the field were 266˚C and 112permil at Kwun Tong and 269˚C and

279permil at Stanley on the first day and 280˚C and 245permil at Kuwn Tong and 300˚C and 234

at Stanley on the fifth day

Data analysis

To determine morphological differences among the bivalves species the ratios of HL WL AB

TB and STI were compared with the one-way analysis of variance (ANOVA) tests To determine

whether Reishia clavigera prey randomly on the three bivalve species (no prey selection) the

analysis was conducted as suggested by Underwood and Clarke (2005) The number of bivalves

eaten in the single choice experiment (stage 1) and the number of bivalves eaten in the multiple

choice experiment (stage 2) were used to derive the maximal likelihood estimators (for more

details of the test see Underwood and Clarke 2005 [29]) Thirty-three random pairs of experi-

mental units of predation on stage 1 (including three bivalve species) and predation on stage 2

were chosen for comparisons The observed number of bivalves eaten was compared with the

expected number using a X2 test with k-1 degree of freedom (k = number of bivalve species)

Predation by native whelk Reishia clavigera on invasive bivalves

PLOS ONE | httpsdoiorg101371journalpone0196578 May 16 2018 5 16

A one-way ANOVA was used to compare the survival among bivalve species (fixed 3 lev-

els) for the non-choice and multiple choice experiments Because none of the bivalves died

under control conditions (without whelks) the control was not included in the analyses A

two-way ANOVA was used to compare the survival among bivalve species (fixed 3 levels) and

salinity (fixed 2 levels) As only one individual of Xenostrobus securis died under 22permil the

control was not included in the data analyses

For the field experiment a two-way ANOVA was conducted to compare survival of the 3

bivalve species (fixed 3 levels) exposed to R clavigera between sites (fixed 2 levels) The sur-

vival of the bivalves was also compared using a two-way ANOVA among the three bivalve spe-

cies (fixed 3 levels) and between the predation treatments (fixed 2 levels open vs control

panels) for each site separately A Tukeyrsquos HSD test was conducted for post-hoc comparison

when factors were significantly different Normality and homoscedasticity of variance were

checked with Shapiro-Wilk tests and Levene tests respectively In cases when the data violated

the homoscedasticity assumption for the ANOVA (in most cases due to 0 or 100 survival in

some datasets) and no transformation was possible we conducted the tests with raw data and

the alpha value was decreased to 001 to reduce Type I error [30]

Results

For morphological features of the bivalve species the results of HL ratios suggested that Myti-lopsis sallei have the roundest shells (Table 1) The results of WL ratios indicated that M salleiand Brachidontes variabilis are more cupped than Xenostrobus securis (Table 1) The results of

ABTB ratios and STI indicated that B variabilis have the largest adductor muscles (propor-

tionally to total biomass) and the thickest shells whereas X securis have the smallest adductor

muscles and thinnest shells among the bivalve species (Table 1)

Twenty out of the 33 X2 test comparisons between observed and expected predation for

stage 1 (single choice) and stage 2 (multiple choice) showed no significant differences

(Table 2) However the 13 remaining comparisons had significant differences The overall

results indicated that in most of the cases the whelk did not show a particular preference for

any of the three bivalve species

In the single choice experiment survival of Xenostrobus securis and Mytilopsis sallei was sig-

nificantly lower than that of Brachidontes variabilis after being exposed for 24-h to Reishia cla-vigera (Table 3A and Fig 2A) Less than 20 of the individuals of X securis and M sallei

Table 1 Summary of morphological features of the bivalve species Xenostrobus securis Mytilopsis sallei and Bra-chidontes variabilis presented as average ratios of maximum shell height (H) and width (W) to length (L) adduc-

tor muscle biomass (AB) to total biomass (TB) and average of shell thickness index (STI) Values in brackets

indicate the range of minimum and maximum values while a bolded P value indicate statistically significant difference

among the three species in the corresponding parameter (by ANOVA tests)

X securis M sallei B variabilis PSize (cm) 1674 1384 1481

(1510ndash1888) (1006ndash1756) (1157ndash1815)

HL 052 058 051 lt0001

(047ndash055) (052ndash065) (048ndash054)

WL 038 043 044 lt0001

(033ndash043) (038ndash047) (041ndash051)

ABTB 0014 0015 0021 0032

(0006ndash0028) (0009ndash0027) (0015ndash0034)

STI 059 065 072 0033

(048ndash076) (050ndash089) (053ndash091)

httpsdoiorg101371journalpone0196578t001

Predation by native whelk Reishia clavigera on invasive bivalves

PLOS ONE | httpsdoiorg101371journalpone0196578 May 16 2018 6 16

survived whereas over 80 of B variabilis survived In the multiple choice experiments the

survival of the bivalves (exposed together with the whelks) showed a similar pattern as that

observed in the single choice experiment Survival of X securis and M sallei was significantly

lower than B variabilis (Table 3B and Fig 2B) Xenostrobus securis and M sallei showed an

average survival of 12 and 37 respectively in contrast to 78 survival in B variabilisMultiple choice experiments under the two salinity conditions showed that the survival of

the bivalve species was significantly affected by the interaction of salinity and bivalve species

Table 2 Results of Chi-square tests to determine prey preference by Reishia clavigera on the bivalves Xenostrobus securis (Xs) Mytilopsis sallei (Ms) and Brachidontesvariabilis (Bv)

Test Obs Obs Obs Obs Obs Obs Exp Exp Exp Exp Exp Exp X2 PXs Ms Bv Xs Ms Bv Xs Ms Bv Xs Ms BvS1 S1 S1 S2 S2 S2 S1 S1 S1 S2 S2 S2

1 12 12 3 6 0 0 120 120 25 27 27 06 735 gt005

2 8 12 3 4 6 0 81 120 22 22 32 06 478 gt010

3 12 12 4 6 5 4 120 120 49 25 25 10 1642 lt0005

4 9 1 1 4 0 0 91 07 07 52 04 04 128 gt010

5 12 11 1 5 4 0 120 110 07 30 28 02 209 gt010

6 8 12 1 3 2 0 83 120 08 24 34 02 104 gt010

7 8 12 3 6 5 1 85 120 27 22 31 07 794 lt005

8 12 12 4 6 4 3 120 120 46 25 25 10 1008 lt0025

9 12 11 1 6 4 0 120 109 07 30 28 02 371 gt010

10 12 12 3 6 6 6 120 120 48 25 25 10 3548 lt0005

11 9 1 1 6 5 0 84 28 05 43 14 03 1145 lt001

12 12 11 1 6 0 0 120 107 08 31 27 02 581 gt010

13 12 12 3 4 6 0 120 120 23 27 27 05 521 gt010

14 12 11 1 6 5 4 120 109 28 28 25 07 2424 lt0005

15 8 12 3 4 0 0 87 120 26 22 31 07 526 gt010

16 9 1 1 5 4 0 85 26 06 44 13 03 710 gt005

17 12 12 4 3 2 0 120 120 35 26 26 08 103 gt010

18 12 12 3 6 5 1 120 120 28 27 27 06 632 gt005

19 8 12 3 6 4 3 84 120 37 21 30 09 1247 lt001

20 12 12 4 6 4 0 120 120 31 27 27 07 584 gt010

21 9 1 1 6 6 6 72 27 27 35 13 13 3947 lt0005

22 12 11 1 6 5 0 120 110 07 30 28 02 496 gt010

23 9 12 3 6 0 0 97 120 25 24 30 06 913 lt005

24 12 12 1 4 6 0 120 120 07 29 29 02 396 gt010

25 12 1 1 6 5 4 120 29 25 41 10 09 3051 lt0005

26 9 11 3 4 0 0 95 108 26 25 28 07 452 gt010

27 8 12 4 5 4 0 85 120 31 22 30 08 510 gt010

28 12 11 3 3 2 0 120 110 26 28 26 06 081 gt010

29 8 12 1 6 5 1 84 120 13 23 33 04 792 lt005

30 12 1 3 6 4 3 120 26 34 40 09 11 1628 lt0005

31 12 12 3 6 4 0 120 120 23 27 27 05 521 gt010

32 9 12 4 6 6 6 90 120 53 20 27 12 3072 lt0005

33 12 11 1 6 5 0 120 110 07 30 28 02 496 gt010

The test compared observed (Obs) and expected (Exp) number of bivalves eaten in single choice (stage 1 = S1) and multiple choice experiments (stage 2 = S2)

Probabilities in bold indicate prey preference

httpsdoiorg101371journalpone0196578t002

Predation by native whelk Reishia clavigera on invasive bivalves

PLOS ONE | httpsdoiorg101371journalpone0196578 May 16 2018 7 16

(Table 3C and Fig 3A) Post hoc analyses indicated that survival of Xenostrobus securis (25)

was significantly lower than Mytilopsis sallei (77) and Brachidontes variabilis (91) under

salinity treatment of 32permil but bivalve species survival did not significantly differ (survival

above 83) under salinity of 22permil (Fig 3)

The results of the acute temperature and salinity test indicated that the survival of Reishiaclavigera was significantly reduced by low salinity (12permil) in particular at 30˚C (Table 3D and

Fig 3B) At 22permil the survival of the whelk was only reduced at 14˚C while no mortality was

observed at 32permil across all temperature treatments

All whelks used in the field experiment survived throughout the experimental period

There were dead bivalves and few missing bivalves on the open panels Possible missing

bivalves were removed by predators because in the control panels all bivalves were still

attached after the exposure period Survival of the bivalves exposed to Reishia clavigera in the

field was significantly different among the three bivalve species (Table 4A and Fig 4A) In gen-

eral Xenostrobus securis had the lowest survival among the bivalve species with 16 survival

at Kwun Tong and 0 at Stanley Mytilopsis sallei had a survival of 60 at Kwun Tong and

36 at Stanley whereas Brachidontes variabilis survival was 70 at Kwun Tong and 33 at

Stanley Though survival tended to be lower at Stanley Pier there was no statistically signifi-

cant difference in the results between the two sites (Table 4A and Fig 4A)

Survival of the bivalves on the open panels exposed to natural predators on fouling commu-

nities did not statistically differ from the control treatments in both Kwun Tong and Stanley

pier (Table 4B and 4C and Fig 4B and 4C) However X securis generally had the lowest aver-

age survival in the open panel treatments among the three bivalve species about 83 in Kwun

Tong and 73 in Stanley pier whereas the survival in control treatments was above 90 for all

the species in both sites

Table 3 Results of the ANOVA tests to compare the survival of the invasive bivalves Xenostrobus securis and

Mytilopsis sallei and the native Brachidontes variabilis exposed to the predatory whelk Reishia clavigera in A) single

choice experiment B) multiple choice experiment and C) multiple choice experiment under two salinities for

24-h and D) survival of Reishia clavigera after the 96-h acute temperature and salinity test

df MS F PA) Single choice

Species 2 14272 1688 lt 0001

Error 15 846

B) Multiple choice

Species 2 4476 1309 lt 0001

Error 30 342

C) Multiple choice under two salinities

Species 2 1908 976 0001

Salinity 1 2178 1114 0002

Sp x Sa 2 869 445 0020

Error 30 196

D) Acute test on R clavigeraTemperature 2 270 348 0053

Salinity 2 781 1005 0001

Temp x Sa 4 337 433 0013

Error 18 078

Acute test analysis was conducted with raw data and alpha value reduced to 001 due to heterogeneity of variances

Bold P-values indicate significant differences

httpsdoiorg101371journalpone0196578t003

Predation by native whelk Reishia clavigera on invasive bivalves

PLOS ONE | httpsdoiorg101371journalpone0196578 May 16 2018 8 16

Predation by native whelk Reishia clavigera on invasive bivalves

PLOS ONE | httpsdoiorg101371journalpone0196578 May 16 2018 9 16

Discussion

The results of this study clearly indicate that both invasive bivalves Xenostrobus securis and

Mytilopsis sallei are more vulnerable than the native Brachidontes variabilis to predation by

the whelk Reishia clavigera Moreover the similar survival of bivalves obtained from both sin-

gle choice and multiple choice experiments indicate that the whelk probably does not have a

preference for any particular bivalve species [29]

The low survival of the invasive species could be caused by a lack of effective defense and

anti-predatory adaptation to the predatory whelk [18 31 32] Anti-predatory responses in

bivalves are diverse and these could vary from changes in size and thickness of the shells [18]

enlargement of the adductor muscle [33] increase of byssal thread production [34] reduction

of clearance rate to lower chemical cues that attract predators [35] and movements and aggre-

gation with other bivalves [36] This predation experiment exposed the bivalves without previ-

ous exposure to the predator and hence did not establish anti-predatory responses Reishiaclavigera attacks bivalves by boring the shell via a chemo-mechanical process [16 17] Based

on our observations R clavigera preyed on bivalves by boring the edge of the valves The shell

thickness index indicated that Xenostrobus securis had the thinner shell followed by Mytilopsissallei and Brachidontes variabilis Consequently R clavigera may spend less time handling the

invasive bivalves which could explain their low survival in the experiments In contrast B var-iabilis has a thicker shell as well as a crenulated valve margin (absent in the invasive bivalves)

that could provide a better defense against the whelks In this case invasive bivalves may

require to thicken their shells to avoid predation by R clavigera

The survival of Reishia clavigera under the acute temperature and salinity test is consistent

with its distribution in Hong Kong [16] As showed in the current study the lowest survival of

R clavigera was under the combination of 12permil and 30˚C Populations could therefore be

drastically affected by prolonged drops in salinity during summer (rainy season) In the pres-

ent survival experiment under two salinity conditions predation on bivalve species decreased

in moderately low salinity treatments where R clavigera likely became stressed under salinity

of 22permil However sub-lethal responses should be measured to corroborate environmental

stress on the whelk in further studies

Studies have found that physical habitats and environmental conditions modify predator-

prey interactions between benthic species [37 38] Recruitment and survival of the invasive

bivalve Musculista senhousia in a California estuary for example is higher under low salinity

where predators cannot access them [4] Therefore the interaction between Reishia clavigeraand the bivalves must be affected by moderate low salinities in the field which could explain

the current distribution of the invasive bivalves in estuarine environments in Hong Kong [21]

As demonstrated in this study Xenostrobus securis would have benefited the most by low

salinity conditions increasing its survival from 25 (at 32permil) to 83 (at 22permil) Low salinities

in summer season could reduce predation pressure on communities inhabited by Reishia clavi-gera Xenostrobus securis larvae which develop normally in salinities of 8ndash17permil [39] may

spread to surrounding areas during low salinity events but their long-term establishment will

depend on predation and competition pressure in those communities Interestingly this study

showed that Mytilopsis sallei had a higher average survival under 32permil in the salinity experi-

ment compared to the survival in the previous single and multiple choice experiments (Figs 1

Fig 2 Survival (plusmn 95 CI) of the invasive bivalves Xenostrobus securis and Mytilopsis sallei alongside the native Brachidontes variabilisexposed to the predatory whelk Reishia clavigera in A) single choice experiment (stage 1) and B) multiple choice experiment (stage 2)

for 24-h Bars with different lowercase letters indicate significantly different mean values (based on the ANOVA and the Tukeyrsquos post-hoc

tests Plt 005)

httpsdoiorg101371journalpone0196578g002

Predation by native whelk Reishia clavigera on invasive bivalves

PLOS ONE | httpsdoiorg101371journalpone0196578 May 16 2018 10 16

Predation by native whelk Reishia clavigera on invasive bivalves

PLOS ONE | httpsdoiorg101371journalpone0196578 May 16 2018 11 16

and 2) This difference could be caused by the static seawater used in the salinity experiment

A previous acute temperature and salinity test on these bivalves indicated that X securis has

higher clearance rate than M sallei [23] High clearance rate is related to a higher release of

chemical cues that attract predators [35] Under static seawater conditions X securis may

attract R clavigera releasing predation pressure on M sallei which could indicate some level

of prey preference

The predation pattern of Reishia clavigera on bivalves is very similar between the laboratory

and field experiments as revealed in this study Consistently Xenostrobus securis had the lowest

average survival compared to Mytilopsis sallei and Brachidontes variabilis Xenostrobus securisseemed to have better survival at the Kwun Tong typhoon shelter than at Stanley pier (Fig 4A)

The lower seawater quality (lower salinity hypoxia sedimentation pollution etc) in Kwun

Tong typhoon shelter [40] likely reduced the predation efficacy of R clavigera

In the present field study the survival of bivalves in open panels exposed to natural preda-

tors did not statistically differ from the controls after 5 days of exposure In contrast a similar

field experiment carried out in an intertidal rocky shore found that the survival of exotic

bivalves decreased to about 30 in 3 days [41] The high abundance of fouling species (ie

high prey availability) on the piers in Hong Kong [42] may have reduced the opportunity or

need of predators to attack the experimental bivalves Predation on fouling communities can

also be lower than on natural reefs [32] Reishia clavigera consumes Brachidontes variabilis but

it also preys on a wide range of other species showing high diet plasticity [17] Hence food

Fig 3 A) Survival of the invasive bivalves to the predatory whelk Reishia clavigera under salinities of 22 and 32permil

for 24-h and B) survival of Reishia clavigera after the 96-h acute temperature and salinity test Error bars

indicate plusmn 95 CI Letters on the columns indicate the results of the Tukeyrsquos post-hoc test for differences among

species within each salinity treatment Numbers on the right corner indicate differences between salinity treatments X

s = Xenostrobus securis

httpsdoiorg101371journalpone0196578g003

Table 4 Results of two-way ANOVA tests to compare the survival of the bivalve species (Xenostrobus securis Mytilop-sis sallei and Brachidontes variabilis) exposed to A) Reishia clavigera at Kwun Tong and Stanley piers and exposed to

natural predators (ie open panels and control panels with predator excluded) in B) Kwun Tong pier and C) Stanley

pier

df MS F PA) Reishia clavigeraSites 1 1469 576 0023

Species 2 1744 684 0004

Si x Sp 2 078 031 0739

Error 30 255

B) Kwun Tong pier

Species 2 144 481 0015

Treatments 1 044 148 0233

Sp x Tr 2 011 037 0694

Error 30 030

C) Stanley pier

Species 2 075 069 0512

Treatments 1 803 734 0011

Sp x Tr 2 019 018 0838

Error 30 109

Tests were conducted with raw data and alpha value reduced to 001 due to heterogeneity of variances The bold P-

value indicates significant differences

httpsdoiorg101371journalpone0196578t004

Predation by native whelk Reishia clavigera on invasive bivalves

PLOS ONE | httpsdoiorg101371journalpone0196578 May 16 2018 12 16

Predation by native whelk Reishia clavigera on invasive bivalves

PLOS ONE | httpsdoiorg101371journalpone0196578 May 16 2018 13 16

availability and preference under natural conditions must be investigated to further under-

stand the role of R clavigera on invasive species

Conclusion

Increasing attention has been given to the use of native predators as biological control agents

for fouling communities and invasive species [10 12] A successful agent species must have life

history traits suitable for the habitat and target invasive species as its prey [43] This study

demonstrated that the whelk Reishia clavigera preyed on the invasive Xenostrobus securis and

Mytilopsis sallei more often than on the native Brachidontes variabilis However this predation

pattern could be due to the predation vulnerability of the invasive bivalves rather than a prey

preference exercised by R clavigera Although R clavigera is a common species on rocky

shores and in fouling communities on piers predation on invasive bivalves is reduced when

salinity decreases Hence our conclusion is that R clavigera may not be the most suitable bio-

control agent for invasive bivalves in Hong Kong marine communities Nevertheless bivalves

have a wide range of predators such as whelks sea stars crabs fishes and birds [34 36 41]

that limit their abundance and distribution Hong Kong as a hotspot of biodiversity [14] has

several species of predatory gastropods and crabs that could prey upon invasive bivalves [16]

in concurrence with R clavigera to control their abundance and distribution

Acknowledgments

The authors would also like to thank the staff of the Swire Institute of Marine Science for their

assistance in this project to Vriko Yu and Ellen Lo for their help with the laboratory work We

are grateful to Dr Nicolas Ory and Sallie Lau for providing comments on an early draft of the

manuscript We also thank Greg Dietl Geerat J Vermeij and an anonymous reviewer for their

constructive feedbacks

Author Contributions

Conceptualization Juan C Astudillo Timothy C Bonebrake Kenneth M Y Leung

Formal analysis Juan C Astudillo

Investigation Juan C Astudillo

Methodology Kenneth M Y Leung

Supervision Timothy C Bonebrake Kenneth M Y Leung

Writing ndash original draft Juan C Astudillo Timothy C Bonebrake Kenneth M Y Leung

References1 Byers JE Competition between two estuarine snails Implications for invasions of exotic species Ecol-

ogy 2000 81(5) 1225ndash39 httpsdoiorg1018900012-9658(2000)081[1225cbtesi]20co

2 Byers JE Competition in marine invasions In Rilov G Crooks JA editors Biological invasions in

marine ecosystems ecological management and geographic perspectives Springer-Verlag Berlin

Heidelberg 2009 pp 245ndash60

Fig 4 Survival (plusmn 95 CI) of bivalve species (Xenostrobus securis Mytilopsis sallei and Brachidontes variabilis)

exposed to A) Reishia clavigera at Kwun Tong and Stanley piers and exposed to natural predators (ie open

panels and control panels with predator excluded) in B) Kwun Tong pier and C) Stanley pier Letters on the right

corner indicate the results of the Tukeyrsquos post-hoc tests between species

httpsdoiorg101371journalpone0196578g004

Predation by native whelk Reishia clavigera on invasive bivalves

PLOS ONE | httpsdoiorg101371journalpone0196578 May 16 2018 14 16

3 Keane RM Crawley MJ Exotic plant invasions and the enemy release hypothesis Trends Ecol Evol

2002 17(4) 164ndash70 httpsdoiorg101016S0169-5347(02)02499-0

4 Cheng BS Hovel KA Biotic resistance to invasion along an estuarine gradient Oecologia 2010 164

(4) 1049ndash59 httpsdoiorg101007s00442-010-1700-7 PMID 20602118

5 Dethier MN Hacker SD Physical factors vs biotic resistance in controlling the invasion of an estuarine

marsh grass Ecol Appl 2005 15(4) 1273ndash83 httpsdoiorg1023074543436

6 Shea K Chesson P Community ecology theory as a framework for biological invasions Trends Ecol

Evol 2002 17(4) 170ndash6 httpsdoiorg101016S0169-5347(02)02495-3

7 Piola RF Johnston EL Pollution reduces native diversity and increases invader dominance in marine

hard-substrate communities Divers Distrib 2008 14(2) 329ndash42 httpsdoiorg101111j1472-4642

200700430x

8 Preisler RK Wasson K Wolff WJ Tyrrell MC Invasions of estuaries vs the adjacent open coast a

global perspective In Rilov G Crooks JA editors Biological invasions in marine ecosystems ecologi-

cal management and geographic perspectives Springer-Verlag Berlin Heidelberg 2009 pp 587ndash

617

9 Atalah J Hopkins GA Forrest BM Augmentative biocontrol in natural marine habitats persistence

spread and non-target effects of the sea urchin Evechinus chloroticus PloS ONE 2013 8(11)

e80365 httpsdoiorg101371journalpone0080365 PMID 24260376

10 Dumont CP Urriago JD Abarca A Gaymer CF Thiel M The native rock shrimp Rhynchocinetes typus

as a biological control of fouling in suspended scallop cultures Aquaculture 2009 292(1ndash2) 74ndash9

httpsdoiorg101016jaquaculture200903044

11 Castilla JC Duran LR Human exclusion from the rocky intertidal zone of central Chile the effects on

Concholepas concholepas (Gastropoda) Oikos 1985 45 391ndash399 httpsdoiorg1023073565575

12 Atalah J Newcombe EM Hopkins GA Forrest BM Potential biocontrol agents for biofouling on artificial

structures Biofouling 2014 30(8) 999ndash1010 httpsdoiorg101080089270142014956734 PMID

25287610

13 Claremont M Vermeij GJ Williams ST Reid DG Global phylogeny and new classification of the Rapa-

ninae (Gastropoda Muricidae) dominant molluscan predators on tropical rocky seashores Mol Phylo-

genet Evol 2013 66(1) 91ndash102 httpsdoiorg101016jympev201209014 PMID 23026810

14 Ng TPT Cheng MCF Ho KKY Lui GCS Leung KMY Williams GA Hong Kongrsquos rich marine biodiver-

sity the unseen wealth of South Chinarsquos megalopolis Biodivers Conser 2017 26(1) 23ndash36 https

doiorg101007s10531-016-1224-5

15 Guo X Zhao D Jung D Li Q Kong LF Ni G et al Phylogeography of the rock shell Thais clavigera

(Mollusca) Evidence for long-distance dispersal in the northwestern Pacific PloS ONE 2015 10(7)

e0129715 httpsdoiorg101371journalpone0129715 PMID 26171966

16 Morton B Morton J The sea shore ecology of Hong Kong Hong Kong Hong Kong University Press

1983

17 Tong LKY The feeding ecology of Thais clavigera and Morula musiva (Gastropoda Muricidae) in Hong

Kong Asian Mar Biol 1986 3 163ndash78

18 Cheung SG Lam S Gao QF Mak KK Shin PKS Induced anti-predator responses of the green mussel

Perna viridis (L) on exposure to the predatory gastropod Thais clavigera Kuster and the swimming

crab Thalamita danae Stimpson Mar Biol 2004 144(4) 675ndash84 httpsdoiorg101007s00227-003-

1233-2

19 Harper E Morton B Muricid predation upon an under-boulder community of epibyssate bivalves in the

Cape DrsquoAguilar Marine Reserve Hong Kong In Morton B editor Proceedings of the eighth interna-

tional marine biological workshop the marine flora and fauna of Hong Kong and southern China Hong

Kong 1995 Hong Kong Hong Kong University Press 1997 pp 263ndash284

20 Taylor JD Morton B The diets of predatory gastropods in the Cape drsquoAguilar Marine Reserve Hong

Kong Asian Mar Biol 1996 13 141ndash66

21 Lai RWS Perkins MJ Ho KKY Astudillo JC Yung MMY Russell BD et al Hong Kongrsquos marine envi-

ronments History challenges and opportunities Regi Stud Mar Sci 2016 8 259ndash273 httpsdoiorg

101016jrsma201609001

22 Morton B Leung KF Introduction of the alien Xenostrobus securis (Bivalvia Mytilidae) into Hong Kong

China Interactions with and impacts upon native species and the earlier introduced Mytilopsis sallei

(Bivalvia Dreissenidae) Mar Pollut Bull 2015 92134ndash142 httpsdoiorg101016jmarpolbul2014

12046 PMID 25599632

23 Astudillo JC Bonebrake TC Leung KM The recently introduced bivalve Xenostrobus securis has

higher thermal and salinity tolerance than the native Brachidontes variabilis and established Mytilopsis

Predation by native whelk Reishia clavigera on invasive bivalves

PLOS ONE | httpsdoiorg101371journalpone0196578 May 16 2018 15 16

sallei Mar Pollut Bull 2017 118 229ndash236 httpsdoiorg101016jmarpolbul201702046 PMID

28259420

24 Morton B Life-history characteristics and sexual strategy of Mytilopsis sallei (Bivalvia Dreissenacea)

introduced into Hong Kong J Zool 1989 219 469ndash85 httpsdoiorg101111j1469-79981989

tb02594x

25 Astudillo JC Wong JCY Dumont CP Bonebrake TC Leung KMY Status of six non-native marine spe-

cies in the coastal environment of Hong Kong 30 years after their first record BioInvasions Rec 2014

3(3) 123ndash37 httpsdoiorg103391bir20143301

26 Veiga P Rubal M Arenas F Incera M Olabarria C Sousa-Pinto I Does Carcinus maenas facilitate the

invasion of Xenostrobus securis J Exp Mar Biol Ecol 2011 406(1) 14ndash20 httpsdoiorg101016j

jembe201105035

27 Seed R Predatorndashprey relationships between the swimming crab Thalamita danae Stimpson (Deca-

poda Portunidae) and the mussels Perna viridis (L) and Brachidontes variabilis (Krauss) In Morton B

editor Proceedings of the second international marine biological workshop the marine flora and fauna

of Hong Kong and southern China Hong Kong Hong Kong University Press 1990 pp 993ndash1013

28 Fitzer SC Vittert L Bowman A Kamenos NA Phoenix VR Cusack M Ocean acidification and temper-

ature increase impact mussel shell shape and thickness problematic for protection Ecology and evolu-

tion 2015 5(21)4875ndash84 httpsdoiorg101002ece31756 PMID 26640667

29 Underwood AJ Clarke KR Solving some statistical problems in analyses of experiments on choices of

food and on associations with habitat J Exp Mar Biol Ecol 2005 318(2) 227ndash37 httpsdoiorg10

1016jjembe200412014

30 Quinn GP Keough MJ Experimental design and data analysis for biologists Cambridge University

Press 2002

31 deRivera CE Ruiz GM Anson HH Jivoff P Biotic resistance to invasion native predator limits abun-

dance and distribution of an introduced crab Ecology 2005 86(12) 3364ndash76 httpsdoiorg101890

05-0479

32 Dumont CP Gaymer CF Thiel M Predation contributes to invasion resistance of benthic communities

against the non-indigenous tunicate Ciona intestinalis Biol Invasions 2011 13(9) 2023ndash2034 https

doiorg101007s10530-011-0018-7

33 Reimer O Tedengren M Phenotypical improvement of morphological defences in the mussel Mytilus

edulis induced by exposure to the predator Asterias rubens Oikos 1996 75(3) 383ndash90 httpsdoi

org1023073545878

34 Caro AU Escobar J Bozinovic F Navarrete SA Castilla JC Phenotypic variability in byssus thread pro-

duction of intertidal mussels induced by predators with different feeding strategies Mar Ecol Prog Ser

2008 372 127ndash34 httpsdoiorg103354meps07701

35 Shin PKS Yang FY Chiu MY Cheung SG Cues from the predator crab Thalamita danae fed different

prey can affect scope for growth in the prey mussel Perna viridis Mar Freshw Behav Phy 2009 42(5)

343ndash55 httpsdoiorg10108010236240903454202

36 Reimer O Tedengren M Predator-induced changes in byssal attachment aggregation and migration in

the blue mussel Mytilus edulis Mar Freshw Behav Phy 1997 30(4) 251ndash66 httpsdoiorg101080

10236249709379029

37 Byers JE Physical habitat attribute mediates biotic resistance to non-indigenous species invasion

Oecologia 2002 130(1) 146ndash56 httpsdoiorg101007s004420100777 PMID 28547019

38 Munari C Mistri M Short-term hypoxia modulates Rapana venosa (Muricidae) prey preference in Adri-

atic lagoons J Exp Mar Biol Ecol 2011 407(2) 166ndash70 httpsdoiorg101016jjembe201106003

39 Wilson BR Survival and reproduction of the mussel Xenostrobus securis (Lam)(Mollusca Bivalvia

Mytilidae) in a Western Australian estuary Part I Salinity tolerance J Nat Hist 1968 2(3) 307ndash328

httpsdoiorg10108000222936800770341

40 EPD Marine water quality in Hong Kong in 2013 Hong Kong Environmental Protection Department

2013 Available from httpwqrcepdgovhkpdfwater-qualityannual-reportMarineReport2013engpdf

41 Lopez MS Coutinho R Ferreira CEL Rilov G Predator-prey interactions in a bioinvasion scenario dif-

ferential predation by native predators on two exotic rocky intertidal bivalves Mar Ecol Prog Ser 2010

403 101ndash12 httpsdoiorg103354meps08409

42 Huang Z Marine biofouling in Hong Kong a review Acta Oceanol Sin 2003 22(3) 467ndash82

43 Epelbaum A Pearce CM Barker DJ Paulson A Therriault TW Susceptibility of non-indigenous ascid-

ian species in British Columbia (Canada) to invertebrate predation Mar Biol 2009 156(6) 1311ndash20

httpsdoiorg101007s00227-009-1172-7

Predation by native whelk Reishia clavigera on invasive bivalves

PLOS ONE | httpsdoiorg101371journalpone0196578 May 16 2018 16 16